Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET. Although not widely appreciated, measured smFRET efficiencies can vary depending on illumination intensity. Self-healing fluorophores offer robust triplet state suppression to enable accurate smFRET measurements under broad illumination regimes.

Scientific facilities generate vast amounts of data, not only large but also diverse and at a fast speed. Additional challenges are posed by the management of users, instruments, report generation, and invoicing. Here we present EMhub, a web platform designed to address these challenges by supporting daily operations of a scientific facility. It allows manipulation of users, instruments, bookings, and project tracking in a simple way. The application was originally inspired by the needs of a CryoEM facility, but it has been general enough that its implementation is extended to other domains. EMhub is implemented in a modular way, allowing the extension of the core functionality. Moreover, different external processes can be connected to the application via a REST API to automate various tasks (e.g. folder creation, user and password generation, etc) and for monitoring on-the-fly data processing independently of the software used. EMhub has been in use in the last few years at the Swedish National CryoEM Facility and more recently also installed in the CryoEM center at the Structural Biology Department at St. Jude Children's Research Hospital. There, a fully automated single particle pipeline has been implemented for on-the-fly data processing and analysis. At St. Jude, the Xray Crystallography Center and the Single-Molecule Imaging Center have already extended the platform to support their data management workflows.

Single-molecule imaging techniques have provided unprecedented insights into functional changes in composition and conformation across diverse biological systems. As with other biophysical methods, single-molecule fluorescence and Förster resonance energy transfer investigations are typically limited to examination of one sample at a time. Consequently, experimental throughput is restricted, and experimental variances are introduced that can obscure functional distinctions in closely related systems. Here, to address these limitations, we introduce parallel rapid exchange single-molecule fluorescence and single-molecule Förster resonance energy transfer to enable simultaneous steady-state and pre-steady-state interrogations of diverse systems. Using this approach, we elucidate the timing of distinct conformational events underpinning β-arrestin1 activation, unmask antibiotic-induced impacts on messenger RNA decoding fidelity and demonstrate that endogenously encoded ribosomal RNA sequence variation modulates antibiotic sensitivity. This generalizable and scalable method promises to broaden the scope and reproducibility of quantitative single-molecule interrogations of biomolecular function. Parallelized single-molecule fluorescence and single-molecule FRET experiments enable quantitative biophysics investigations of molecular function from multiple samples in a single experiment.

Most scientific facilities produce large amounts of heterogeneous data at a rapid pace. Managing users, instruments, reports and invoices presents additional challenges. To address these challenges, EMhub, a web platform designed to support the daily operations and record-keeping of a scientific facility, has been introduced. EMhub enables the easy management of user information, instruments, bookings and projects. The application was initially developed to meet the needs of a cryoEM facility, but its functionality and adaptability have proven to be broad enough to be extended to other data-generating centers. The expansion of EMHub is enabled by the modular nature of its core functionalities. The application allows external processes to be connected via a REST API, automating tasks such as folder creation, user and password generation, and the execution of real-time data-processing pipelines. EMhub has been used for several years at the Swedish National CryoEM Facility and has been installed in the CryoEM center at the Structural Biology Department at St. Jude Children's Research Hospital. A fully automated single-particle pipeline has been implemented for on-the-fly data processing and analysis. At St. Jude, the X-Ray Crystallography Center and the Single-Molecule Imaging Center have already expanded the platform to support their operational and data-management workflows.

Integral membrane proteins (IMPs) play central roles in cellular physiology and represent the majority of known drug targets. Single-molecule fluorescence and fluorescence resonance energy transfer (FRET) methods have recently emerged as valuable tools for investigating structure-function relationships in IMPs. This review focuses on the practical foundations required for examining polytopic IMP function using single-molecule FRET (smFRET) and provides an overview of the technical and conceptual frameworks emerging from this area of investigation. In this context, we highlight the utility of smFRET methods to reveal transient conformational states critical to IMP function and the use of smFRET data to guide structural and drug mechanism-of-action investigations. We also identify frontiers where progress is likely to be paramount to advancing the field.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes(1). Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure(2,3). Electrophysiological properties of CFTR have been analysed for decades(4-6). The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.

Single-molecule fluorescence studies enable real-time observation of interactions between molecular machines using minuscule amounts of (potentially) precious samples. Experimental repetition is required for optimizations, to test different samples or conditions, and ultimately to perform technical and biological repeats for publication. Here we developed a multiplexed approach that allows us to significantly increase experimental sensitivity, reproducibility, and throughput of single-molecule fluorescence and fluorescence energy transfer imaging. By coupling this approach with nucleic acid barcoding, microfluidic handling systems, and advanced data processing, we aim to automate and simplify multiperspective single-molecule studies to accelerate the emerging field of dynamic structural biology.

β-arrestins bind G protein-coupled receptors (GPCRs) to terminate G-protein signaling and to facilitate other downstream signaling pathways. During activation, β-arrestin's carboxy-terminal tail must disengage from the amino-terminal domain, but little is known about this process. Here, we use single-molecule fluorescence resonance energy transfer imaging of β-arrestin1, alone and in complex with activating phosphorylated receptor carboxy-terminal tail mimetics or full-length receptor, to explore β-arrestin tail dynamics.

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

SARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. S is synthesized as a precursor, processed into S1 and S2 by furin proteases, and activated for fusion when human angiotensin-converting enzyme 2 (hACE2) engages the receptor-binding domain (RBD) and when the N-terminus of S2 is proteolytically processed. Structures of soluble ectodomains and native virus particles have revealed distinct conformations of S, including a closed trimer with all RBD oriented downward, trimers with one or two RBDs up, and hACE2-stabilized conformations with up to three RBD oriented up. Real-time information that connects these structures, however, has been lacking. Here we apply single-molecule Forster Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens into the hACE2-bound S conformation through at least one on-path intermediate, with trypsin partially activating S. Conformational preferences of convalescent patient plasma and monoclonal antibodies suggest mechanisms of neutralization involving either direct competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and on conformations for immunogen design.

The HIV-1 envelope glycoprotein (Env) trimer, composed of gp120 and gp41 subunits, mediates viral entry into cells. Recombinant Env trimers have been studied structurally, but characterization of Env embedded in intact virus membranes has been limited to low resolution. Here, we deploy cryo-electron tomography and subtomogram averaging to determine the structures of Env trimers on aldrithiol-2 (AT-2)-inactivated virions in ligand-free, antibody-bound and CD4-bound forms at subnanometer resolution. Tomographic reconstructions document molecular features consistent with high-resolution structures of engineered soluble and detergent-solubilized Env trimers. One of three conformational states previously predicted by smFRET was not observed by cryo-ET, potentially owing to AT-2 inactivation. We did observe Env trimers to open in situ in response to CD4 binding, with an outward movement of gp120-variable loops and an extension of a critical gp41 helix. Overall features of Env trimer embedded in AT-2-treated virions appear well-represented by current engineered trimers. Cryo-ET and subtomogram averaging analyses are used to determine structures of HIV-1 Env trimers on the surface of inactivated virions.

Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer-based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting "molecular memory."

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4+) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated "SOS" (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing conditions may enable structural characterization of the state 1 conformation of HIV-1 Env.IMPORTANCE The HIV-1 envelope glycoprotein (Env) opens in response to receptor CD4 binding from a pretriggered (state 1) conformation through a necessary intermediate to the three-CD4-bound conformation. The application of smFRET to test the conformational state of existing Env constructs and ligand complexes used for high-resolution structures recently revealed that they correspond to the downstream conformations. The structure of the pretriggered Env conformation, preferentially recognized by broadly neutralizing antibodies, remains unknown. Here, we identify experimental conditions that stabilize membrane-bound and shedding-resistant virus Env trimers in state 1, potentially facilitating structural characterization of this unknown conformational state.

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na + /substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L -Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na + binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins.

Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic(1-8.) Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)(8-10). It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP. 664 trimers)(5,11-18). Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1(JR-FL) strain in complex with the antibody PGT151(19). Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pretriggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

Prevailing dogma holds that ribosomes are uniform in composition and function. Single-molecule investigations of translation reveal, however, that the ribosome and translation factors visit metastable, transient intermediates that can be highly sensitive to even modest perturbations, including small-molecule drugs and single-nucleotide substitutions in ribosomal RNA (rRNA). These insights suggest the possibility that subtle changes in the ribosome's composition may have functional impacts. Recent evidence further implicates changes in the ribosome's core protein composition with gene-specific changes in translational efficiency. In this light, we set out to examine the potential physiological impacts of endogenously encoded sequence variations in the rRNA components of the assembled ribosome. Mammalian genomes encode hundreds of ribosomal DNA operons (rDNA) that exhibit extensive sequence variation with the rRNA components of the ribosome, which are both conserved and expressed in a tissue-specific fashion. Using E.coli as a genetically tractable model system, we now show that nutrient limitation-induced stress changes the relative expression of rDNA operons to alter the ribosomal RNA (rRNA) composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA gene, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes alter the levels of the RpoS sigma factor, the master regulator of the general stress response, to affect the expression of functionally coherent gene sets. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility, and are regulated by ribosome-associated stress response proteins. Specific aspects of these phenotypic differences could be reconstituted in vitro using highly purified translation components. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology.